Separation of Nicotinic Acid and Six Metabolites within 60 Seconds Using High-Flow Gradient Chromatography on Silica Column with Tandem Mass Spectrometric Detection

Summary

Separation of nicotinic acid and its six metabolites (nicotinamide, nicotinuric acid, 1-methy-2-pyridone-5-carboxamide, 1-methy-4-pyridone-5-carboxamide, 1-methyl-nicotinamide, and nicotinamide-N-oxide) within 60 seconds was achieved by using a gradient chromatography at a flow rate of 4 mL min⁻¹ on silica column and aqueous/organic mobile phases with tandem mass spectrometric detection. The fourth metabolite, 1-methyl-4-pyridone-5-carboxamide, did not show isotopic or cross channel interference, even though it co-eluted with nicotinamide-N-oxide. This method was validated to analyze all seven compounds in human plasma and urine samples which were extracted with Waters Oasis MCX 96-well plate. The linearity of the method is 10 to 2000 ng mL⁻¹ for each analyte. The relative standard deviation and relative error for the standards were less than 17% and 8.4%, respectively. The relative standard deviation and relative error for the quality control samples were less than 12% and 13% respectively.